Elderly humans have the highest prevalence of being overweight and obese, suggesting a defect in energy balance. Under physiological conditions, the predominant fuel of choice in the fasted state is non-esterified fatty acids (NEFA). However in aging, NEFA oxidation in the fasted state is impaired and there is increased glucose utilization, suggesting a defect in mitochondrial b-oxidation. Mitochondria rely on antioxidants fo protection against oxidative damage, and glutathione (GSH) is the most abundant endogenous antioxidant. In past and ongoing studies, we observed that elderly humans were deficient in GSH and this was associated with a 50% lower fat oxidation and 65% higher plasma NEFA levels compared to younger humans. We found that GSH deficiency in elderly humans occurred due to decreased synthesis, because of decreased availability of the GSH precursor amino acids cysteine and glycine. Supplementing the diet of these elderly subjects with cysteine and glycine for 2-weeks fully restored GSH synthesis and concentrations, and significantly lowered markers of cellular damage due to oxidative stress to levels found in young controls. After GSH restoration, fasted NEFA oxidation increased by 30%, and fasted plasma FFA fell by 36%, suggesting that impaired NEFA oxidation in aging is a reversible defect. To investigate whether GSH deficiency in aging is linked to impaired NEFA oxidation and predisposes to excess accumulation of body and tissue fat, we studied and found that 80-week old mice had significantly lower GSH in skeletal muscle and liver, impaired whole-body NEFA oxidation, and higher total body- and intrahepatic- fat, compared to young 20-week old mice. Supplementing diets of older mice with cysteine and glycine for 6-weeks corrected GSH deficiency in muscle and liver, restored whole-body NEFA oxidation, and lowered total body fat, hepatic fat and body weight. These data suggest that in aging, GSH deficiency predisposes to impaired NEFA oxidation and excess body and tissue fat; correcting GSH deficiency by supplementing cysteine and glycine in the diet restores NEFA oxidation and reverses these defects. This proposal will identify mechanisms linking GSH deficiency to impaired mitochondrial NEFA oxidation in elderly humans, and test a simple, safe, inexpensive therapy based on altering dietary composition by supplementing GSH precursor amino-acids cysteine and glycine to correct GSH deficiency and restore mitochondrial NEFA oxidation. The proposal will also test whether restoration of GSH and NEFA oxidation will result in loss of body fat, intrahepatic fat and intramyocellular fat in elderly humans. PUBLIC HEALTH RELEVANCE: Mitochondria are the engines of our bodies where the food we eat is burned to make energy, and glutathione is an important protein that protects mitochondria from harm. Elderly humans make very little glutathione, and their mitochondria are not able to burn fat well, and so fat gets stored in fat cells, liver and muscle. This study will help us understand how glutathione helps mitochondria burn fat, and if feeding elderly people with more building block proteins to make and normalize glutathione will help mitochondria burn fat correctly, and cause them to lose weight and have less fat in their liver and muscle.